【作者】 宋荣峰；

【导师】 张焜和；

【作者基本信息】 江西医学院 ， 内科学消化， 2003， 硕士

【摘要】 Objective Serosa metastatic carcinomas usually cause malignant serous cavity effusions. The differentiation between malignant and benign effusions is sometimes difficult but very important in clinical practice. Laboratory tests are essential to the differentiation, but current available tests can not solve the problem satisfactorily. Ras gene is an important proto-oncogene, and its mutations are frequently found in many malignant tumors. Detecting ras gene mutations and expression in effusions is probably useful in differentiating malignant effusions from benign ones. In the present study, point mutations of codon 12 of K-ras and H-ras gene in the effusion supernatants were detected by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and P21ras expressions in the effusion exfoliative cells were detected by immunocytochemistry, and their values in diagnosis of malignant effusions were evaluated.Methods Fresh specimens of serous cavity effusions were obtained. Exfoliative cells and supernatants were collected after centrifugation, respectively. Two cell smears were prepared for Papanicolaou staining and cytological diagnosis, by which the specimens were divided into benign and malignant effusions. Genome DNA in the supernatants was conventionally extracted by proteinase K digestion followed by phenol/chloroform extraction. The DNA fragments containing codon 12 of K-ras and H-ras gene were amplified by PCR, respectively, and were cleaved by restriction endonuclease Mva I or Msp I, respectively. Digestive products were purified and identified by<WP=6>means of polyacrylamide gel electrophoresis and silver staining. On the other hand, some of exfoliative cells were processed with a "standardized" procedure: removing red cells (when existing), fixed in 1% paraformaldehyde, resuspended in a solution of 1% BSA-PBS, adjusting the cell concentration to 2×106 cells per milliliter, and finally cell smears were prepared for immunocytochemistry. The primary antibody was monoclonal mouse anti-human P21ras, the second antibody was the biotinylated goat anti-mouse IgG. Streptavidin labeled with horseradish peroxidase was as the enzyme system. The color was developed with DAB. Results were observed under a light microscope. Results A total of 108 specimens of serous cavity effusions were collected, including 53 cases of malignant effusions and 55 cases of benign effusions. PCR amplification of codon 12 of K-ras and H-ras genes was successful in 101 supernatants of the specimens (93.5%). In 52 malignant specimens, the codon 12 mutation rates of K-ras and H-ras gene were 55.8% and 30.8%, respectively. In 49 benign specimens, the mutation rates were 10.2% and 4.1%. The differences of the mutation rates of K-ras or H-ras were significant between benign and malignant effusions (P<0.001). The specificities (Sp) of K-ras and H-ras gene mutation for the diagnosis of malignant effusions were 89.8% and 95.9%, the positive predictive values (PV+) were 85.3% and 88.9%, the negative predictive values (PV-) were 65.8% and 56.6%, and the diagnostic accordance rates (DAR) were 72.3% and 62.4%.For the immunocytochemical staining of P21ras, tumor exfoliative cells in 39 of 53 cases (73.6%) of malignant effusions were positive, and most of them expressed strongly. The positive results were observed in 12 of 55 cases (21.8%) of benign effusions, and most of them expressed weakly. The differences of positive rates and expression intensity of P21ras were significant between malignant and benign effusions (P<0.001). Sp of P21ras immunocytochemistry in diagnosis of malignant effusions was 78.2%, PV+ 76.5%, PV 75.4% and DAR 75.9%.Combination analysis of ras gene mutation and P21ras immunocytochemical<WP=7>staining in a parallel and series way, sensitivities for the diagnosis of malignant effusions were 88.7% and 49.1%, Sp 69.1% and 96.4%, PV+ 73.4% and 92.9%, PV- 86.4% and 63.9%, DAR 78.7% and 73.2%. Parallel combination analysis enhances the sensitivity and series combination analysis enhances the s更多还原

【Abstract】 Objective Serosa metastatic carcinomas usually cause malignant serous cavity effusions. The differentiation between malignant and benign effusions is sometimes difficult but very important in clinical practice. Laboratory tests are essential to the differentiation, but current available tests can not solve the problem satisfactorily. Ras gene is an important proto-oncogene, and its mutations are frequently found in many malignant tumors. Detecting ras gene mutations and expression in effusions is probably useful in differentiating malignant effusions from benign ones. In the present study, point mutations of codon 12 of K-ras and H-ras gene in the effusion supernatants were detected by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and P21ras expressions in the effusion exfoliative cells were detected by immunocytochemistry, and their values in diagnosis of malignant effusions were evaluated.Methods Fresh specimens of serous cavity effusions were obtained. Exfoliative cells and supernatants were collected after centrifugation, respectively. Two cell smears were prepared for Papanicolaou staining and cytological diagnosis, by which the specimens were divided into benign and malignant effusions. Genome DNA in the supernatants was conventionally extracted by proteinase K digestion followed by phenol/chloroform extraction. The DNA fragments containing codon 12 of K-ras and H-ras gene were amplified by PCR, respectively, and were cleaved by restriction endonuclease Mva I or Msp I, respectively. Digestive products were purified and identified by<WP=6>means of polyacrylamide gel electrophoresis and silver staining. On the other hand, some of exfoliative cells were processed with a "standardized" procedure: removing red cells (when existing), fixed in 1% paraformaldehyde, resuspended in a solution of 1% BSA-PBS, adjusting the cell concentration to 2×106 cells per milliliter, and finally cell smears were prepared for immunocytochemistry. The primary antibody was monoclonal mouse anti-human P21ras, the second antibody was the biotinylated goat anti-mouse IgG. Streptavidin labeled with horseradish peroxidase was as the enzyme system. The color was developed with DAB. Results were observed under a light microscope. Results A total of 108 specimens of serous cavity effusions were collected, including 53 cases of malignant effusions and 55 cases of benign effusions. PCR amplification of codon 12 of K-ras and H-ras genes was successful in 101 supernatants of the specimens (93.5%). In 52 malignant specimens, the codon 12 mutation rates of K-ras and H-ras gene were 55.8% and 30.8%, respectively. In 49 benign specimens, the mutation rates were 10.2% and 4.1%. The differences of the mutation rates of K-ras or H-ras were significant between benign and malignant effusions (P<0.001). The specificities (Sp) of K-ras and H-ras gene mutation for the diagnosis of malignant effusions were 89.8% and 95.9%, the positive predictive values (PV+) were 85.3% and 88.9%, the negative predictive values (PV-) were 65.8% and 56.6%, and the diagnostic accordance rates (DAR) were 72.3% and 62.4%.For the immunocytochemical staining of P21ras, tumor exfoliative cells in 39 of 53 cases (73.6%) of malignant effusions were positive, and most of them expressed strongly. The positive results were observed in 12 of 55 cases (21.8%) of benign effusions, and most of them expressed weakly. The differences of positive rates and expression intensity of P21ras were significant between malignant and benign effusions (P<0.001). Sp of P21ras immunocytochemistry in diagnosis of malignant effusions was 78.2%, PV+ 76.5%, PV 75.4% and DAR 75.9%.Combination analysis of ras gene mutation and P21ras immunocytochemical<WP=7>staining in a parallel and series way, sensitivities for the diagnosis of malignant effusions were 88.7% and 49.1%, Sp 69.1% and 96.4%, PV+ 73.4% and 92.9%, PV- 86.4% and 63.9%, DAR 78.7% and 73.2%. Parallel combination analysis enhances the sensitivity and series combination analysis enhances the s更多还原